Intra-platform comparison of 25-mer and 60-mer oligonucleotide Nimblegen DNA microarrays.

BACKGROUND: We performed a Nimblegen intra-platform microarray comparison by assessing two categories of flax target probes (short 25-mers oligonucleotides and long 60-mers oligonucleotides) in identical conditions of target production, design, labelling, hybridization, image analyses, and data filtering. We compared technical parameters of array hybridizations, precision and accuracy as well as specific gene expression profiles. RESULTS: Comparison of the hybridization quality, precision and accuracy of expression measurements, as well as an interpretation of differential gene expression in flax tissues were performed. Both array types yielded reproducible, accurate and comparable data that are coherent for expression measurements and identification of differentially expressed genes. 60-mers arrays gave higher hybridization efficiencies and therefore were more sensitive allowing the detection of a higher number of unigenes involved in the same biological process and/or belonging to the same multigene family. CONCLUSION: The two flax arrays provide a good resolution of expressed functions; however the 60-mers arrays are more sensitive and provide a more in-depth coverage of candidate genes potentially involved in different biological processes.

Identification of cell wall proteins in the flax (Linum usitatissimum) stem.

Sequential salt (CaCl2 , LiCl) extractions were used to obtain fractions enriched in cell wall proteins (CWPs) from the stem of 60-day-old flax (Linum usitatissimum) plants. High-resolution FT-ICR MS analysis and the use of recently published genomic data allowed the identification of 11 912 peptides corresponding to a total of 1418 different proteins. Subcellular localization using TargetP, Predotar, and WoLF PSORT led to the identification of 152 putative flax CWPs that were classified into nine different functional classes previously established for Arabidopsis thaliana. Examination of different functional classes revealed the presence of a number of proteins known to be involved in, or potentially involved in cell-wall metabolism in plants. The flax stem cell wall proteome was also compared with transcriptomic data previously obtained on comparable samples. This study represents a major contribution to the identification of CWPs in flax and will lead to a better understanding of cell wall biology in this species.

Identification and characterization of miRNAs and their potential targets in flax.

MicroRNAs (miRNAs) are small non-protein coding regulatory RNAs released after the cleavage of a primary transcript. A computational homology search of expressed sequence tags (ESTs) available in public databases allowed the identification of 20 conserved miRNAs belonging to 13 different families in flax (Linum usitatissimum). Most of the miRNAs were 21 nucleotides-long and carried a uracil at the 5' end. They originated from precursor transcripts that vary greatly in length. A single precursor containing 2 different stem-loop structures, each one carrying a member of the miR398 family, was identified for the first time in plants. qRT-PCR analyses of 4 selected miRNAs indicated that all were differentially expressed in flax tissues. The 20 miRNAs could potentially regulate 112 different targets including genes involved in cell wall metabolism. Analyses of pri-miRNA and potential gene expression profiles in a publically available microarray data set allowed the identification of a number of highly opposite pri-miRNA/target gene profiles potentially involved in regulating plantacyanin levels, F-box mediated signalling processes, protein metabolism and ion homeostasis, as well as 6 unknown processes.

Natural hypolignification is associated with extensive oligolignol accumulation in flax stems.

Flax (Linum usitatissimum) stems contain cells showing contrasting cell wall structure: lignified in inner stem xylem tissue and hypolignified in outer stem bast fibers. We hypothesized that stem hypolignification should be associated with extensive phenolic accumulation and used metabolomics and transcriptomics to characterize these two tissues. (1)H nuclear magnetic resonance clearly distinguished inner and outer stem tissues and identified different primary and secondary metabolites, including coniferin and p-coumaryl alcohol glucoside. Ultrahigh-performance liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry aromatic profiling (lignomics) identified 81 phenolic compounds, of which 65 were identified, to our knowledge, for the first time in flax and 11 for the first time in higher plants. Both aglycone forms and glycosides of monolignols, lignin oligomers, and (neo)lignans were identified in both inner and outer stem tissues, with a preponderance of glycosides in the hypolignified outer stem, indicating the existence of a complex monolignol metabolism. The presence of coniferin-containing secondary metabolites suggested that coniferyl alcohol, in addition to being used in lignin and (neo)lignan formation, was also utilized in a third, partially uncharacterized metabolic pathway. Hypolignification of bast fibers in outer stem tissues was correlated with the low transcript abundance of monolignol biosynthetic genes, laccase genes, and certain peroxidase genes, suggesting that flax hypolignification is transcriptionally regulated. Transcripts of the key lignan genes Pinoresinol-Lariciresinol Reductase and Phenylcoumaran Benzylic Ether Reductase were also highly abundant in flax inner stem tissues. Expression profiling allowed the identification of NAC (NAM, ATAF1/2, CUC2) and MYB transcription factors that are likely involved in regulating both monolignol production and polymerization as well as (neo)lignan production.

Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray.

BACKGROUND: Flax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax. RESULTS: Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties. CONCLUSION: All results suggest that our high-density flax oligo-microarray platform can be used as a very sensitive tool for analyzing gene expression in a large variety of tissues as well as in different cultivars. Moreover, this highly reliable platform can also be used for the quantification of mRNA transcriptional profiling in different flax tissues.

Populations of weedy crop-wild hybrid beets show contrasting variation in mating system and population genetic structure.

Reproductive traits are key parameters for the evolution of invasiveness in weedy crop-wild hybrids. In Beta vulgaris, cultivated beets hybridize with their wild relatives in the seed production areas, giving rise to crop-wild hybrid weed beets. We investigated the genetic structure, the variation in first-year flowering and the variation in mating system among weed beet populations occurring within sugar beet production fields. No spatial genetic structure was found for first-year populations composed of F1 crop-wild hybrid beets. In contrast, populations composed of backcrossed weed beets emerging from the seed bank showed a strong isolation-by-distance pattern. Whereas gametophytic self-incompatibility prevents selfing in wild beet populations, all studied weed beet populations had a mixed-mating system, plausibly because of the introgression of the crop-derived Sf gene that disrupts self-incompatibility. No significant relationship between outcrossing rate and local weed beet density was found, suggesting no trends for a shift in the mating system because of environmental effects. We further reveal that increased invasiveness of weed beets may stem from positive selection on first-year flowering induction depending on the B gene inherited from the wild. Finally, we discuss the practical and applied consequences of our findings for crop-weed management.

Fine-scale geographical structure of genetic diversity in inland wild beet populations.

Introgression arising from crop-to-wild gene flow provides novel sources of genetic variation in plant species complexes. Hybridization within the Beta vulgaris species complex is of immediate concern; crop lineages (B. vulgaris ssp. vulgaris) hybridize easily with their wild relatives (B. vulgaris ssp. maritima) thereby threatening wild beet gene diversity with genetic swamping. Hybridization 'hotspots' occur in European seed production areas because inland ruderal wild beets occur and reproduce in sympatry with cultivated beets. We studied gene flow occurring between seed-producing cultivars and ruderal wild B. vulgaris in southwestern France to determine whether feral beets, arising from unharvested cultivated seed, represent an opportunity for crop-to-wild gene flow. We surveyed 42 inland ruderal beet populations located near seed production fields for nucleo-cytoplasmic variation and used a cytoplasmic marker diagnostic of cultivated lines. Occurrence of cultivated-type cytoplasm within ruderal populations clearly reflected events of crop seed escape. However, we found no genetic signatures of nuclear cultivated gene introgression, which suggests past introgression of cultivated cytoplasm into a wild nuclear background through seed escape rather than recent direct pollen flow. Overall, patterns of genetic structure suggested that inland ruderal wild beet populations act as a metapopulation, with founding events involving a few sib groups, followed by low rates of seed or pollen gene flow after populations are established. Altogether, our results indicate that a long-lived seed bank plays a key role in maintaining cultivated-type cytoplasm in the wild and highlight the need for careful management of seed production areas where wild and cultivated relatives co-occur.

Nuclear and cytoplasmic genetic diversity in weed beet and sugar beet accessions compared to wild relatives: new insights into the genetic relationships within the Beta vulgaris complex species.

Hybridization between cultivated species and their wild relatives is now widely considered to be common. In the Beta vulgaris complex, the sugar beet seed multiplication areas have been the scene of inadvertent pollination of sugar beet seed bearers by wild ruderal pollen donors, generating a weedy form of beet which infests sugar beet fields in European countries. Up to now, investigations of evolutionary dynamics of genetic diversity within the B. vulgaris complex were addressed using few genetical markers and few accessions. In this study, we tackled this issue using a panel of complementary markers: five nuclear microsatellite loci, four mitochondrial minisatellite loci and one chloroplastic PCR-RFLP marker. We sampled 1,640 individuals that illustrate the actual distribution of inland ruderal beets of South Western France, weed beets and wild sea beets of northern France as well as the diversity of 35 contemporary European diploid cultivars. Nuclear genetic diversity in weed beets appeared to be as high as those of ruderal beets and sea beets, whereas the narrowness of cultivar accessions was confirmed. This genetic bottleneck in cultivars is even more important in the cytoplasmic genome as only one haplotype was found among all sugar beet cultivars. The large majority of weed beet populations also presented this unique cytoplasmic haplotype, as expected owing to their maternal cultivated origin. Nonetheless, various cytoplasmic haplotypes were found within three populations of weed beets, implying wild-to-weed seed flows. Finally, our findings gave new insights into the genetical relationships between the components of the B. vulgaris complex: (1) we found a very strong genetic divergence between wild sea beet and other relatives, which was unexpected given the recent evolutionary history and the full cross-compatibility of all taxa and (2) we definitely confirmed that the classification into cultivated, wild, ruderal and weed forms according to their geographical location, phenotype or their domesticated status is clearly in accordance with genetic clustering despite the very recent domestication process of sugar beet.

Long distance pollen-mediated gene flow at a landscape level: the weed beet as a case study.

Gene flow is a crucial parameter that can affect the organization of genetic diversity in plant species. It has important implications in terms of conservation of genetic resources and of gene exchanges between crop to wild relatives and within crop species complex. In the Beta vulgaris complex, hybridization between crop and wild beets in seed production areas is well documented and the role of the ensuing hybrids, weed beets, as bridges towards wild forms in sugar beet production areas have been shown. Indeed, in contrast to cultivated beets that are bi-annual, weed beets can bolt, flower and reproduce in the same crop season. Nonetheless, the extent of pollen gene dispersal through weedy lineages remains unknown. In this study, the focus is directed towards weed-to-weed gene flow, and we report the results of a pollen-dispersal analysis within an agricultural landscape composed of five sugar beet fields with different levels of infestation by weed beets. Our results, based on paternity analysis of 3240 progenies from 135 maternal plants using 10 microsatellite loci, clearly demonstrate that even if weedy plants are mostly pollinated by individuals from the same field, some mating events occur between weed beets situated several kilometres apart (up to 9.6 km), with rates of interfield-detected paternities ranging from 11.3% to 17.5%. Moreover, we show that pollen flow appears to be more restricted when individuals are aggregated as most mating events occurred only for short-distance classes. The best-fit dispersal curves were fat-tailed geometric functions for populations exhibiting low densities of weed beets and thin-tailed Weibull function for fields with weed beet high densities. Thus, weed beet populations characterized by low density with geographically isolated individuals may be difficult to detect but are likely to act as pollen traps for pollen emitted by close and remote fields. Hence, it appears evident that interfield pollen-mediated gene flow between weed beets is almost unavoidable and could contribute to the diffusion of (trans)genes in the agricultural landscape.

Emergence of gynodioecy in wild beet (Beta vulgaris ssp. maritima L.): a genealogical approach using chloroplastic nucleotide sequences.

Gynodioecy is a breeding system where both hermaphroditic and female individuals coexist within plant populations. This dimorphism is the result of a genomic interaction between maternally inherited cytoplasmic male sterility (CMS) genes and bi-parentally inherited nuclear male fertility restorers. As opposed to other gynodioecious species, where every cytoplasm seems to be associated with male sterility, wild beet Beta vulgaris ssp. maritima exhibits a minority of sterilizing cytoplasms among numerous non-sterilizing ones. Many studies on population genetics have explored the molecular diversity of different CMS cytoplasms, but questions remain concerning their evolutionary dynamics. In this paper we report one of the first investigations on phylogenetic relationships between CMS and non-CMS lineages. We investigated the phylogenetic relationships between 35 individuals exhibiting different mitochondrial haplotypes. Relying on the high linkage disequilibrium between chloroplastic and mitochondrial genomes, we chose to analyse the nucleotide sequence diversity of three chloroplastic fragments (trnK intron, trnD-trnT and trnL-trnF intergenic spacers). Nucleotide diversity appeared to be low, suggesting a recent bottleneck during the evolutionary history of B. vulgaris ssp. maritima. Statistical parsimony analyses revealed a star-like genealogy and showed that sterilizing haplotypes all belong to different lineages derived from an ancestral non-sterilizing cytoplasm. These results suggest a rapid evolution of male sterility in this taxon. The emergence of gynodioecy in wild beet is confronted with theoretical expectations, describing either gynodioecy dynamics as the maintenance of CMS factors through balancing selection or as a constant turnover of new CMSs.

Spatial genetic structure within a metallicolous population of Arabidopsis halleri, a clonal, self-incompatible and heavy-metal-tolerant species.

Arabidopsis halleri, a close wild relative of A. thaliana, is a clonal, insect-pollinated herb tolerant to heavy metals (Zn, Pd, Cd) and a hyperaccumulator of Zn and Cd. It is of particular interest in the study of evolutionary processes and phytoremediation. However, little is known about its population gene flow patterns and the structure of its genetic diversity. We used five microsatellite loci to investigate the genetic structure at a fine spatial scale (10 cm to 500 m) in a metallicolous population of A. halleri. We also studied the contributions made by clonal propagation and sexual reproduction (seed and pollen dispersal) to the genetic patterns. Clonal diversity was high (D(G) > 0.9). Clonal spread occurs only at short distances (< 1 m). Both clonal spread and limited dispersal, associated with sexual reproduction, contribute to the significant spatial genetic structure revealed by spatial autocorrelation analysis. The shape of the autocorrelogram suggests that seed dispersal is restricted and pollen flow extensive, which may be related to intense activity by insect pollinators. Clonal spread was more extensive in the lowly polluted zone than in the highly polluted zone. This cannot be interpreted as a strategy for promoting the propagation of adapted genotypes under the harshest ecological constraints (highest heavy metal concentrations). The higher fine-scale spatial genetic structure found in the lowly polluted zone can be ascribed to plant densities that were lower than in the highly polluted zone. No evidence of genetic divergence due to spatial heavy metal heterogeneity was found between lowly and highly polluted zones.